帕金森病(PD)的发病率随着年龄的增加而迅速上升。随着全球老龄化的到来,PD患者的数量随着老年人口的增加而增加,尤其是在中国。以前,我们发现益肾破痰汤(YCD),根据临床经验开出处方,有缓解症状的潜力,延缓进展,控制PD的发展。尽管如此,潜在的机制作用还有待探索。
■这项研究通过网络药理学和实验验证的系统方法检查了YCD在缓解PD方面的可能治疗作用,旨在为中医中医管理提供新的认识。
■YCD的化学结构和性质来自中药系统药理学数据库(TCMSP),Swissadme,PubChem,和PubMed。YCD和PD的潜在目标是使用瑞士目标预测确定的,GeneCard,PubChem,还有UniProt.草药成分目标网络是通过Cytoscape软件创建的。此外,通过使用STRING数据库,筛选蛋白质-蛋白质相互作用(PPI)网络。基因功能GO和KEGG途径富集分析通过Metascape数据库进行。制备SD大鼠YCD含药血清,和SH-SY5Y细胞用鱼藤酮预处理以建立PD模型。研究YCD对这些细胞的影响,并探讨p38丝裂原活化蛋白激酶(MAPK)通路的作用机制。用血清或p38MAPK途径抑制剂预处理细胞。本研究采用细胞计数试剂盒(CCK)-8测定和Hoechst33,342染色来评估YCD含药大鼠血清在鱼藤酮处理的SH-SY5Y细胞上诱导的生存力和形态学变化。通过流式细胞术评估细胞凋亡。免疫荧光染色评估微管相关蛋白2(MAP2)水平。采用酶联免疫吸附试验(ELISA)定量检测炎症介质白细胞介素-1β(IL-1β)的浓度,白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)。此外,测定活性氧(ROS)和超氧化物歧化酶(SOD)水平。蛋白质印迹法测量总和磷酸-p38MAPK(p-p38)的表达。
■这项研究确定了YCD中的65种活性成分,发现靶向801个特定基因。通过筛选,从PD和YCD之间的172个重叠靶标池中鉴定出63个潜在的核心靶标。通过GO和KEGG分析检查了这些靶标,揭示了它们与MAPK的实质性相关性,PI3K-Akt信号通路,积极控制蛋白质磷酸化,和神经退行性疾病的途径。SH-SY5Y细胞用2μM鱼藤酮处理48小时,将细胞活力降低到50%,并降低MAP2表达,增加细胞凋亡率,氧化应激,炎症,和p-p38表达式。含药YCD的大鼠血清显着提高了活力,降低细胞凋亡率,并增加MAP2的表达。YCD含药血清增加SOD,降低ROS并抑制IL-6,IL-1β和TNF-α水平,从而抑制鱼藤酮处理的SH-SY5Y细胞的氧化应激和炎症。此外,含药YCD的血清大大降低了鱼藤酮诱导的p-p38表达。SB203580,p38MAPK的特异性抑制剂,还可以抑制p-p38的表达,凋亡,恢复细胞的形态损伤,还可以改善炎症和氧化应激。
■YCD增强细胞活力,降低细胞凋亡率,炎症,和体外氧化应激。这些有益作用可能涉及抑制p38途径和抑制p38MAPK的磷酸化。
The incidence of Parkinson\'s disease (PD) rises rapidly with the increase of age. With the advent of global aging, the number of patients with PD is rising along with the elderly population, especially in China. Previously, we found that Yishen chuchan decoction (YCD), prescribed based on clinical experience, has the potential of alleviating symptoms, delaying the progression, and controlling the development of PD. Nonetheless, the underlying mechanistic role is yet to be explored.
UNASSIGNED: This research examined the possible therapeutic effects of YCD in alleviating PD via a systematic approach with network pharmacology and experimental validation, aiming at providing a new understanding of traditional Chinese medicine management regarding PD.
UNASSIGNED: The chemical structure and properties of YCD were adopted from Traditional Chinese Medicine System Pharmacology Database (TCMSP), SwissADME, PubChem, and PubMed. The potential targets for YCD and PD were identified using Swiss Target Prediction, GeneCard, PubChem, and UniProt. The herbal-component-target network was created via the Cytoscape software. Moreover, by using the STRING database, the protein-protein interaction (PPI) network was screened. Gene function GO and KEGG pathway enrichment analyses were performed via the Metascape database. YCD-medicated Rat Serum from Sprague-Dawley (SD) Rats was prepared, and SH-SY5Y cells were preconditioned with rotenone to develop the PD model. To examine the impact of YCD on these cells and explore the mechanistic role of the p38 mitogen-activated protein kinase (MAPK) pathway, the cells were pretreated with either serum or a p38 MAPK pathway inhibitor. This study employed the Cell Counting Kit (CCK)-8 assay and Hoechst 33,342 staining to evaluate the viability and morphological changes induced by the YCD-medicated rat serum on rotenone-treated SH-SY5Y cells. Apoptosis was assessed by Flow cytometry. Immunofluorescence staining assessed the microtubule-associated protein 2 (MAP2) level. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the concentrations of inflammatory mediators interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Also, reactive oxygen species (ROS) and superoxide dismutase (SOD) levels were determined. Western Blotting measured the expression of total and phospho-p38 MAPK (p-p38).
UNASSIGNED: This study identified 65 active components in YCD, which were found to target 801 specific genes. By screening, 63 potential core targets were identified from a pool of 172 overlapping targets between PD and YCD. These targets were examined by GO and KEGG analyses revealing their substantial correlation to MAPK, PI3K-Akt signaling pathways, positively controlling protein phosphorylation, and pathways of neurodegenerative diseases. SH-SY5Y cells were treated with 2 μM rotenone for 48 h, which reduced cell viability to 50 %, and reduced MAP2 expression, increased the rate of apoptosis, oxidative stress, inflammation, and p-p38 expressions. YCD-medicated rat serum significantly improved the viability, reduced the apoptosis rate, and increased the MAP2 expression. YCD-medicated serum increased SOD, reduced ROS and suppressed IL-6, IL-1β and TNF-α levels, thus inhibiting oxidative stress and inflammation in rotenone-treated SH-SY5Y cells. Moreover, YCD-medicated serum substantially lowered the p-p38 expression induced by rotenone. SB203580, a specific inhibitor of p38 MAPK, could also inhibit the p-p38 expression, apoptosis, and restore morphological damage of cells, also improve inflammation and oxidative stress.
UNASSIGNED: YCD enhanced cell viability and reduced apoptosis rate, inflammation, and oxidative stress in vitro. These beneficial effects could potentially involve the suppression of p38 pathway and suppressed the phosphorylation of p38 MAPK.